Technique for acceleration of apoptotic cell death

ABSTRACT

A first embodiment of a cell culture system has a cell death accelerator comprising one or more cell death inducing substances, including serum albumin, hemoglobin, glycine and glutamic acid. In a second embodiment a cell death inhibitor comprises one or more kinds of cell death inhibiting substances which include mercapto group containing amino acids, other mercapto group containing compounds and tryptophan. In a third embodiment a cell death inhibitor comprises an inhibitor of RNA or protein synthesis, optionally augmented with a thiol. The system can be applied to selectively induce death of cultured cells, such as neoplastic cell lines, or to inhibit death of other cells, such as neoplastic cell lines or non-neoplastic cells such as brain cells.

This is a divisional of application Ser. No. 08/510,017 filed on Aug. 1,1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

This invention relates to a cell death accelerator for inducing celldeath and a cell death inhibitor for inhibiting cell death.

2. Description of the Related Art.

For the purpose of culturing cells in vitro, serum is generally added toa synthetic culture medium in a concentration range of about 10-20%. Themedium is a pH-balanced salt solution containing various nutrients suchas vitamins, amino acids, and sugars. When serum or plasma is highlyconcentrated in the medium, beyond this range however, cell death isinduced.

The cell death-inducing activity is seen in all the sera or plasma sofar tested regardless of source (species or age), or heat treatment toinactivate complement. Two types of cell death are now generallyrecognized. The first is passive, resulting from lack of oxygen,mechanical crush injury, or other extreme change in the extracellularenvironment. The second type of cell death is called apoptosis, anactive energy-requiring intracellular process that culminates in DNAfragmentation and osmotic lysis of the cell. The latter is an autonomousphysiological death sometimes referred to as programmed cell death, andit occurs normally during development of the nervous system, skin, andother epithelial organs. Morphologically, apoptosis is characterized byblebbing of the plasma membrane and nuclear condensation. These visiblechanges are accompanied by synthesis of a specific protein(s) includingnuclease followed by fragmentation of chromosomal DNA Severalapoptosis-related genes have already been identified in the nematode C.elegans and in mammalian lymphocytes.

Apoptosis is now the focus of much attention, because it appears to becrucial for normal tissue and organogenesis, neural development, andimmune reactions to foreign antigens including those borne by infectiousviruses.

The serum contains some components, i.e., proteins such as albumin andglobulin, salts such as NaCl, KCl and CaCl₂, vitamins, more than twentyamino acids, such as glycine, cystine, cysteine, alanine and tryptophan,and other components. Heretofore adequate investigation of theindividual functions of each component in the serum has not been carriedout. The present inventors disclosed that amino acids, such as cystineand cysteine, when added to the serum, decrease the toxic effect of theserum and accelerates growth and/or multiplication of cultured cells inJapanese Patent Application No. 270719/1990 filed on Oct. 9, 1990. Atpresent, however, investigation of the toxic factor itself has not beencarried out at all, and the characterization of components which caninhibit the toxicity has been insufficient. In the noted JapaneseApplication, the inventors disclosed that cysteine and cystine promotedcell growth and/or multiplication, and that tryptophan can prevent celldeath.

Recently, Evan et al. (1992) reported that cultured fibroblasts diedwhen the mitotic cycle was interrupted during c-myc expression.Serum-induced cell death may involve a similar mechanism. Cells enteringthe mitotic cycle in response to an excess of growth signals from serummay die after interruption of the cycle by thiol deficiency. Previously,we reported that a cell-death-inducing activity was present in a lowmolecular weight fraction of serum (M.W.<1,000) ( Kurita and Namiki,1993a). Subsequently, it was found that this activity was inhibited bythiols. It was water-soluble, heat-resistant, and had charcoal affinity.Approximate molecular weight of the factor was approximately 100-200dalton upon size-sieve HPLC. In addition to low molecular weightfactors, traces of macromolecules in the fraction are now thought to benecessary for cell death. The latter may act as a death signal, andc-myc may be also involved in the signal transduction. Further studiesare needed to address these issues. Nevertheless, serum-induced celldeath appears to be a type of apoptosis resulting from a disturbance inthiol metabolism.

SUMMARY OF THE INVENTION

Under the above mentioned circumstances, we have investigated thefunctions of individual components in the serum, and have achieved thepresent invention on the basis of the results of the investigation.

It is an object of the present invention to provide a cell deathaccelerator for inducing cell death.

It is another object of the present invention to provide a cell deathinhibitor for inhibiting cell death.

According to a first aspect of the invention, a cell death agentcomprises at least one cell death inducing substance which is selectedfrom serum albumin, hemoglobin, glycine and glutamic acid to be added toblood. Cell death of cultured cells is produced by adding one or more ofthe above mentioned substances to a culture medium containing serum. Forexample, the cell death accelerator of the invention can be applied totreat cancer because the cell death inducing substance destroys cancercells or inhibits growth and/or multiplication of cancer cells.

According to a second aspect of the invention, a cell death inhibitorcomprises at least one cell death inhibiting substance which is selectedfrom mercapto group containing amino acids such as cysteine, cystine andso forth, other mercapto group containing compounds and tryptophan, theselected substance to be added to blood.

The cell death inhibiting substances in the serum are mercapto groupcontaining amino acids, other mercapto group containing compounds andtryptophan. When one or more of the above mentioned cell deathinhibitors are added to a culture medium containing serum, theinhibitors counteract the toxic effects of the toxic factor in theserum, and, thereby the cell death of cultured cells is inhibited. Forexample, if the cell death inhibiting substances are added to aninstillation injection, and then injected into the central nervoussystem of a patient, and directly supplied to a diseased part duringtreatment of brain apoplexy, eye ground apoplexy and so forth, death ofthe patient's cells can be prevented efficiently.

We had previously found that various sera supplemented with amino acidsand vitamins in quantities equivalent to those in basal tissue culturemedium inhibited cell growth but did not induce cell death. Herein wedisclose the identity of the rescue factors as L-cysteine or L-cystineand L-tryptophan, and we examine the effects of thiol-containingmolecules other than L-cysteine on serum induced cell death. All of themwere protective in varying degrees. Death is also prevented by severalinhibitors of protein and RNA synthesis. Interestingly,N-acetyl-L-cysteine (NAC) was as protective as other thiols, but it didnot inhibit uptake of L- ³⁵ S!methionine, suggesting that thiol bearingmolecules were not acting as inhibitors of protein or RNA synthesis.Treatment of cells cultured in FBS with NAC prevented a reduction intheir thiol content, but protein and RNA synthesis inhibitors had nocorresponding effect. In addition, we also demonstrated DNAfragmentation prior to the breakdown of the plasma membrane. Thesefindings suggest that serum-induced cell death represents athiol-mediated apoptosis.

The invention provides a cell system which includes a concentration of alow molecular weight fraction of serum. A population of living cells areexposed to the serum, and optionally to a concentrated and refinedmolecular species that prevents induction of death of the cells by wholeserum. The cells can be human fetal lung fibroblasts, human epithelioidcarcinoma cells, or mouse melanoma cells.

According to an aspect of the invention the molecular species is a thiolor a dithiol homo-dimer thereof. The molecular species can be at leastone of the group consisting of cysteine, glutathione, dithiothreitol,2-mercaptoethanol, dithionite, thioglycolic acid, DL-homocystine,N-acetyl-L-cysteine, 5,5'-dithiobis(2-nitrobenzoic acid), a homo-dimerthereof, and a mixed disulfide thereof.

Preferably the molecular species has a concentration of between about 1and10 mM.

The invention provides a cell system which includes a concentration ofserum. A population of living cells are exposed to the serum, forexample fetal bovine serum, and to a concentrated and refined molecularspecies that accelerates induction of death of the cells. The molecularspecies is selected from the group consisting of serum albumin,hemoglobin, glycine and glutamic acid. The cells can be human fetal lungfibroblasts, human epithelioid carcinoma cells, or mouse melanoma cells.

The invention provides a method of preventing cell death in a cellsystem which is accomplished by providing a concentration of a lowmolecular weight fraction of serum, which is equivalent to a cytotoxicconcentration of whole serum, the latter being sufficient to induce celldeath, exposing a population of living cells to the low molecular weightserum fraction, and optionally exposing the living cells to aconcentrated and refined molecular species that prevents induction ofdeath of the cells by the serum. The cells can be human fetal lungfibroblasts, human epithelioid carcinoma cells, or mouse melanoma cells.According to an aspect of the invention the molecular species is a thiolor a dithiol homo-dimer thereof. The molecular species can be at leastone of the group consisting of cysteine, glutathione, dithiothreitol,2-mercaptoethanol, dithionite, thioglycolic acid, DL-homocystine,N-acetyl-L-cysteine, 5,5'-dithiobis(2-nitrobenzoic acid), a homo-dimerthereof, and a mixed disulfide thereof.

Preferably the molecular species has a concentration of between about 1and 10 mM.

The invention provides a method of preventing serum-induced apoptoticcell death, which is accomplished by providing a cell culture mediumwhich has a concentration of a low molecular weight fraction of serum,which is equivalent to a cytotoxic concentration of whole serum, thelatter being sufficient to induce cell death, enriching the medium withan amino acid selected from the group consisting of L-cysteine,L-cystine, and L-tryptophan, and culturing a cell line in the enrichedmedium.

According to an aspect of the invention the amino acid is L-cysteine ina concentration of between about 0.1 mM and 5 mM.

In another aspect of the invention the amino acid is L-cystine in aconcentration of between about 0.05 mM and 0.5 mM.

The invention provides a method of preventing serum-induced apoptoticcell death, which is performed by providing a cell culture medium has acytotoxic concentration of serum, enriching the medium with a substanceselected from the group consisting of puromycin hydrochloride, emetinehydrochloride, cycloheximide, actinomycin D, ethidium bromide,L-tryptophan, and D-tryptophan, and culturing a cell line in theenriched medium.

According to an aspect of the invention the medium is enriched with athiol, which can be N-acetyl-cysteine.

The invention provides a method of preventing serum-induced apoptoticcell death, which is performed by providing a cell culture medium havinga cytotoxic concentration of serum, culturing a cell line in theenriched medium, and while the cells are growing in culture, inhibitingprotein synthesis or RNA synthesis in the cell line.

BRIEF DESCRIPTION OF THE DRAWING

For a better understanding of these and other objects of the presentinvention, reference is made to the detailed description of theinvention, by way of example, which is to be read in conjunction withthe following drawings, wherein:

FIG. 1 is a chart showing the cell death inhibiting effects ofsubstances on cultured cells;

FIG. 2 is a chart showing the cell death inducing effects of substanceson cultured cells;

FIG. 3 is a chart indicating the effect of serum concentration on cellgrowth;

FIG. 4 is a chart illustrating rescue from serum-induced cell death byamino acids;

FIG. 5 illustrates rescue from serum-induced cell death by inhibitors ofprotein and RNA synthesis;

FIG. 6 is a chart illustrating the effect of protein and RNA synthesisinhibitors on methionine uptake in cultured cells;

FIGS. 7a and 7b are charts respectively illustrating total thiol contentand ethanol-soluble thiol content of cultured cells following incubationin the presence of various reagents;

FIG. 8 is a chart illustrating the relationship of live cultured cellsto time in an experiment on serum toxicity; and

FIGS. 9a and 9b are schematic illustrations of two models ofserum-induced cell death.

DESCRIPTION OF THE PREFERRED EMBODIMENT Abbreviations

As used herein certain abbreviations and acronyms have the followingmeanings:

    ______________________________________                                        ACT D  actinomycin D FBS     fetal bovine serum                               CHX    cycloheximide GSH     glutathione reduced                              CYS    cysteine      GSSG    glutathione oxidized                             CYS-CYS                                                                              cystine       2-ME    2-mercaptoethanol                                DL-HC  DL-homocystine                                                                              NAC     N-acetyl-L-cysteine                              DTN    sodium dithionite                                                                           NON     non-inhibitor                                    DTNB   5,5'dithiobis(2-                                                                            PBS     Phosphate buffered saline                               nitrobenzoic acid)                                                                          PMC     puromycin.2HCl                                   DTT    dithiothreitol                                                                              TGA     thioglycolic acid                                EAA    essential amino acids                                                                       MEM     minimal essential medium                         EMT    emetine.2HCl  NEAA    nonessential amino acids                         ET Br  ethidium bromide                                                       ______________________________________                                    

In one experiment, a cell system had a cytotoxic concentration of serumsufficient to induce cell death. A population of living cells wasexposed to the serum, and a concentrated and refined molecular speciesthat prevents induction of death of the cells by the serum was includedin a culture medium. Suitable cells were human fetal lung fibroblasts,human epithelioid carcinoma cells, and mouse melanoma cells. Moreparticularly TIG-cells, i.e., human fetal lung fibroblasts, were seededinto a 96-well multiplate (1×10⁴ cells/well) and incubated overnight.Thereafter the culture medium was removed to be exchanged with testmedium, a culture medium containing fetal bovine serum (FBS) and variousselected reagents. Six days after the exchange of the culture medium,living cells in the culture medium were counted using a dye elutionmethod to obtain the living cell density associated with each reagent.

The molecular species used was a thiol or a dithiol homo-dimer thereof,or a mixed disulfide in various concentrations which included the rangeof between about 1 and 10 mM.

The following reagents were used: L-cystine (L-CYSCYS);D-cystine(D-CYSCYS); L-cysteine (L-CYS); D-cysteine (D-CYS); reducedglutathione (GSH); oxidized glutathione (GSSG); DL-homocystine (DL-HC);N-acetyl-L-cysteine (NAC); 2-mercaptoethanol (2-ME); thioglycolic acid(TG); dithiothreitol (DTT); 5-5,5'-dithiobis(2-nitro benzoic acid)(DTNB); sodium dithionite (DTN); and L-tryptophan (L-TRP). Theconcentration of each reagent was set at four levels: 0.01 mM, 0.1 mM,1.0 mM and 10 mM.

A result of the experience is shown in FIG. 1. In FIG. 1, each reagentis indicated along the abscissa and the corresponding cell density isindicated along the ordinate. The height of each column in the graphindicates the mean of the measured cell densities of six wells, and abar on the top of each column, if shown, indicates the standard error.

According to the result, reagents which had a mercapto group in theirchemical structures conduced to inhibition of the cell death. L-CYSCYS,D-CYSCYS, L-CYS, 2-ME, TG and DTN showed greatly inhibiting effects.They could prevent cell death even at a concentration of I mM. It wasrecognized that mercapto group containing amino acids and other mercaptogroup containing compounds could inhibit cell death.

Experiments on cell death inducing substances which exist in serum willnow be explained. A cell system was used which had a cytotoxicconcentration of serum sufficient to induce cell death. A population ofliving cells was exposed to the serum, and the culture medium containeda concentrated and refined molecular species that accelerates inductionof death of the cells by the serum. The molecular species was one of thegroup consisting of serum albumin, hemoglobin, glycine and glutamicacid. Suitable cells were human fetal lung fibroblasts, humanepithelioid carcinoma cells, and mouse melanoma cells.

A low molecular weight fraction in FBS serum was freeze-dried, and asolvent then added. The insoluble fraction in was removed bycentrifugation to purify the low molecular weight fraction. The solublefraction thereby obtained was freeze-dried again, and washed inacetonitrile. The resulting freeze-dried substance was dissolved indistilled water to prepare a sample solution having an approximately20-fold dilution of the low molecular weight fraction with respect toFBS, supposing that recovery of the low molecular weight fractiontherefrom is 100%. Thereafter, the sample solution was filtered througha filter having a 0.2μ mesh.

This sample solution was added to Hank's solution so as to obtain a testmedium containing 10 volume % of above mentioned sample solution, 1volume % of FBS and 10 volume % of MEM. Further, each reagent orcombination of the reagent and cysteine being tested was added to thetest medium at reagent concentrations of 1 mM and 10 mM.

TIG-cells were seeded into a 24-well multiplate (5×10³ cells/well)having the test medium and incubated for 48 hours. Thereafter livingcell density was measured using the dye elution method.

According to the experimental results, glycine, glutamic acid andtryptophan showed a cell death inducing effect at 1 mM. Phenylalanine,aspartic acid, asparagine and glutamine also showed a cell deathinducing effect at 10 mM. On the other side, thyrosin, serine andglycylglycine did not show a cell death inducing effect, but inhibitedgrowth and/or multiplication of cells. It was furthermore proved thathemoglobin had growth and/or multiplication inhibiting effect in thesame manner.

These and other experiments will now be disclosed in further detail.

Cell cultures

Human fetal lung fibroblasts (TIG-1), human epithelioid carcinoma (HeLa)cells, and mouse melanoma (B16) cells were obtained from the JapaneseCancer Research Resources Bank. All cells were maintained in Eagle's MEMcontaining 10% FBS at 37° C. Cell cultures were examined andphotographed with a Nikon Diaphot phase-contrast inverted microscope.

Reagents

Amino acids: L-cysteine or L-cystine (Kanto Chemical), D-cysteine(Sigma), D-cystine (Wako), L-tryptophan (Kanto), D-tryptophan,DL-homocystine (Wako), glutathione oxidized form (Sigma grade III),reduced form (Merck), dithiothreitol (Wako), 2-mercaptoethanol,thioglycolic acid, N-acetyl-L-cysteine, sodium dithionite (Kanto),5,5'-dithiobis(2-nitro benzoic acid) (Kanto), emetine.2HCl (Fluka AG.),cycloheximide, puromycin. 2HCl, actinomycin D, ethidium bromide (Sigma).

Serum and media

FBS was from Boehringer Mannheim (lot. 614413, 562044, 147013). Eagle'sMEM "Nissui" was from Nissui Pharmaceutical Co., EAA (essential aminoacids) supplement for MEM×50, NEM (nonessential amino acids) supplementfor MEM×100 and vitamin supplement for MEM×100 were from BoehringerMannheim, Flow Laboratories, and Dainippon Pharmaceutical Co.,respectively.

Ultrafiltration of FBS

FBS was filtered through an ultrafiltration membrane YM2 (M.W.1,000)(Amicon Co.). FBS was concentrated tenfold and diafiltered with atenfold volume of deionized water to remove the low-molecular-weightfraction. The resultant macromolecular fraction of FBS was againconcentrated tenfold and was diluted to the original FBS volume with10/9 concentrated Eagle's MEM. pH and osmolality were adjusted to7.2±0.2 and 290±10 mosmol/Kg.H₂ O), as the low-molecular-weight-fractiondepleted FBS.

Automated cell counting

Appropriate numbers of cells were seeded into 96-well culture platescontaining Eagle's MEM, 10% FBS (100 μl/well) (Corning). After overnightincubation at 37° C. in 5% CO₂, the culture medium was removed. Wellswere washed with calcium and magnesium-free PBS (CMF-PBS), and 100 μl oftest medium was added. After several days of incubation, cells wereharvested by trypsinization and suspended in Isotone II (CoulterElectronics). Cell number was measured with a Coulter Counter ZM(Coulter Electronics) and displayed numbers were corrected with standardhemocytes. We previously verified that the numbers displayed on thecounter were in agreement with those counted using a hemocytometer.Results were expressed as the means (±SE) of six independentmeasurements.

Cell counting by dye elution method

TIG-1 cells were seeded in 96-well microplates in 100 μl of Eagle's MEM,10% FBS. After several day's incubation, medium was removed and thecells were washed with CMF-PBS. Cell quantity was determined by themethod of Hori et al. (1988) with a minor modification. Cells werestained with 50 μl of 0.5% crystal violet per well in ethanol/water(1:4) for 10 min and then washed with water. The dye was eluted with 150μl of 33% acetic acid per well, and the absorbance at 600 nm wasmeasured by a microplate reader (CORONA MTP-22). Results were expressedas the means (±SE) of six independent determinations. We preliminarilyconfirmed that the absorbance at 600 nm was proportional to the densityof TIG-1 cells cultured in MEM, 10% FBS.

Cell viability

TIG-1 cells were seeded in 48-well microplates in 100 μl of Eagle's MEM,10% FBS. After incubation, cells were harvested with 0.1% trypsin, inCMF-PBS, and 0.5% trypan blue in PBS was added. Cells that were notstained were counted with a hemocytometer. Results were expressed as themeans (±SE) of four independent determinations.

L- ³⁵ S!methionine Uptake

TIG-1 cells were seeded into a 24-well multiplate at 1/2 confluency(about ×10⁵ cells/well) and incubated overnight. The next morning, theculture medium was removed and changed to test medium (20 μCi L- ³⁵!methionine (Amersham), 20% CMF-PBS, 80% FBS). After appropriate time(0-16 hr) of incubation, each culture was washed three times with 0.02%EDTA CMF-PBS, and the cells were lysed with 1% SDS. Macromolecules wereprecipitated with each 10 μl TCA and centrifuged, and the precipitatewas washed with 10% TCA. Then, it was dissolved in 300 μl of 1% SDS, 100μl of which was dissolved in 1 ml of liquid scintillation cocktail andcounted (Aloka LSC-700).

Thiol Content Assays

TIG-1 cells were cultured in a 24-well plate (3×10⁵ cells/well) in freshmedium for 24 hr. The medium was then changed to the test medium, MEMplus 10% FBS, Hank's balanced solution, or whole FBS supplemented withvarious reagents. Total thiol content was determined as follows. Afterappropriate hr (0-16) of incubation, cells were washed twice with 0.02%EDTA, CMF-PBS, and they were lysed with 200 μl/well of 1% SDS, 0.02%EDTA, 50 mM Tris-HCl (pH.8.2). The lysate was dyed with 10 μl of 20 mMDTNB dissolved in methanol. Pre-chilled ethanol (600 μl) was added toprecipitate the macromolecules, and precipitates were collected bycentrifugation at 13,000×g for 20 min. Absorbance at 415 nm of thesupernatant was measured by a microplate reader (Bio Rad 3550).Concentration of thiol was calculated by comparison to a GSH standard.Ethanol-soluble thiol was measured as follows. After appropriate hr(0-16) of incubation, cells were washed twice with 0.02% EDTA, CMF-PBS,and they were lysed with 100 μl/well of 1% SDS, 0.02% EDTA, 50 mMTris-HCl (pH.8.2). Macromolecules were precipitated with 300 μl ofpre-chilled ethanol, and precipitates were removed by centrifugation at13,000×g for 20 min. The supernatant was dyed with 10 μl of 20 mM DTNBdissolved in methanol, and the absorbance at 415 nm of the supernatantwas measured by a microplate reader. Concentration of thiol wascalculated by comparison to a GSH standard. Thiol content was expressedas SH mol/living cell number.

DNA Fragmentation Assay

TIG-1 cells (3.5×10⁵ cells/35 mm dish) were washed twice with CMF-PBSand lysed in 0.8 ml 0.6% TE buffer with 1 μg/ml Rnase. Then, 200 μl of5M NaCl was added to the solution and stored at 4° C. overnight, andmacromolecular DNA was pelleted by centrifugation at 13,000×g for 30min. DNA in the supernatant was purified by phenol-chloroformextraction, and traces of phenol were removed by extracting twice withchloroform. Purified DNA was collected by centrifugation at 13,000×g for20 min with 3M potassium acetate and 70% ethanol. Precipitated DNA wasdissolved in TE buffer, and 1/5 to 1/3 volume of the solution waselectrophoresed in a 2.6% agarose gel. Gel was stained by 0.5 μg/mlethidium bromide and photographed in 254 nm ultraviolet.

We previously discovered that most types of cultured cells died inconcentrations of serum exceeding 60%. However, low molecular weightfraction-depleted serum whose osmotic pressure was adjusted with Eagle'sMEM (UF-FBS) never showed cytotoxicity. The low molecular weightfraction thus filtered was similar to MEM in salt content, but it wasdeficient in nutrients such as vitamins and amino acids. We thereforeenriched FBS with these nutrients equivalent to the amounts present inMEM for cell culture.

FIG. 3 shows the effect of serum concentration on TIG-1 cell growth,wherein □ indicates Unenriched FBS; ▴ indicates FBS+AA VIT; and ◯indicates low molecular weight fraction-depleted FBS. In developing thedata illustrated in FIG. 3, TIG-1 cells were seeded at a density of5×10³ cells/well in 96-well microplates in Eagle's MEM containing 10%FBS. After overnight incubation, medium was replaced with Eagle's MEMcontaining several concentrations of FBS. After 6 days, cells harvestedby trypsinization were counted with a Coulter counter.

As shown in FIG. 3, cells died at a high concentration (at least 60%) ofunenriched FBS (Whole FBS), but not in the UF-FBS supplemented with MEM.In addition, cell growth was suppressed in enriched FBS as theconcentration was increased. Nevertheless, the cells remained viable,suggesting MEM contained molecules that prevented cell death. Among thevitamins and amino acids tested, only the latter showed this rescueeffect. One half to fourfold MEM equivalent of amino acids rescued TIG-1cells from serum-induced cell death (data not shown). We measured theconcentration of each amino acid in FBS. Among essential amino acids,L-cysteine or L-cystine and arginine were of less concentration in FBSas compared to those in MEM, which were 8-15 vs. 24 and 1-6 vs. 126,respectively. To identify the amino acids responsible for the rescueeffect, each of the 20 L-amino acids involved in protein synthesis andL-hydroxyproline were tested (data not shown).

Rescue from serum-induced cell death by amino acids is illustrated inFIG. 4. In developing the data illustrated in FIG. 4 TIG-1 cells wereseeded in 96-well microplates at a density of 5×10³ cells/well inEagle's MEM containing 10% FBS. After overnight incubation, medium wasreplaced with FBS containing amino acids. After 6 days, cells werecounted with a Coulter counter. Only two amino acids, L-cysteine orL-cystine and L-tryptophan, showed ability to rescue cells fromserum-induced toxicity (FIG. 3) With L-cysteine, cell death wascompletely prevented at 0.1 mM, and cells grew well at I mM. WithL-cystine, whose maximum solubility is ˜0.5 mM, protective effect wereobserved beginning at 0.05 mM (0.1 mM cysteine equivalent). In contrast,L-tryptophan was effective only at concentrations above 5 mM, and itonly partially rescued the cells. Concentrations of L-cysteine higherthan 5 mM sometimes inhibited cell growth. Similar to TIG-1 cells, HeLacells and B16 cells were also protected by L-cysteine or L-cystine andL-tryptophan (data not shown).

The rate of glutathione synthesis in cultured fibroblasts is known todepend on the cystine content of the medium (Meister and Tate, 1976),and when glutathione was added to the medium of TIG-1 cells, death wasprevented. This also occurred when oxidized glutathione (GSSG) wastested. There was no evidence of extracellular enzymatic reduction ofGSSG or its transport into the cell. Accordingly, the rescue activity ofglutathione may not be due to its ability to conjugate to othermolecules via thiol linkages.

To further study the roles of cysteine and glutathione, experiments wereperformed as before using reducing agents, dithiols, L-amino acids,denaturants, and an active oxygen-eliminating enzyme. These reagents arelisted below. Reducing agents: L-ascorbic acid, L-ascorbic acidphosphate, Mg, DL-α-tocopherol, dithiothreitol, 2-mercaptoethanol,thioglycolic acid, N-acetyl-L-cysteine, sodium dithionite, potassiumferrocyanide, thiourea. Dithiols: DL-homocysteine,5,5'-dithiobis(2-nitro benzoic acid); L-amino acids: L-tryptophan,L-methionine; chelates: ethylene diamine tetra-acetic acid and ethyleneglycol bis(2-aminoethylether). Denaturants: urea, sodium dodecylsulfate; Active-oxygen eliminating enzyme: bovine erythrocyte superoxidedismutase (SOD) (WAKO).

FIG. 1. illustrates rescue from serum-induced cell death by thiols. Indeveloping the data shown in FIG. 1, TIG-1 cells were seeded in 96-wellmicroplates at a density of 1×10⁴ cells/well in Eagle's MEM containing10% FBS. After overnight incubation, medium was removed, and 80 μl ofFBS plus 20 μl of PBS containing cysteine (CYS), cystine (CYS-CYS),glutathione oxidized (GSSG), glutathione reduced (GSH), dithiothreitol(DTT), 2-mercaptoethanol (2-ME), sodium dithionite (DTN), thioglycolicacid (TGA), DL-homocystine (DL-HC), N-acetyl-L-cysteine (NAC), or5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were added to each well. Allreagents were tested for rescue from cell death at concentrationsranging from 10 μM to 1 mM, in some cases to 10 mM. D- and L-cystinewere tested in the range of 1 μM to 100 μM, because of low solubility.After 4 days, cell number was determined by the dye elution method.Reagents were tested in concentrations from 1 pM to 1 mM or 1 μM to 10mM, except in the case of SOD, which was tested in the range 10¹ 10⁴units/ml. Only rescue activity-positive data from independentexperiments are depicted in FIG. 1. All reagents bearing SH or cleaveddithiols possessed the rescue activity, and cysteamine and cystaminealso were effective in preventingcell death. In contrast, thenonthiol-reducing agents were inactive (data not shown).

To determine whether death was due to apoptosis or necrosis, variousinhibitors of protein synthesis and RNA synthesis were tested for therescue activity, since it is generally accepted that in apoptosisspecific protein(s) are synthesized. The procedure is explained withreference to FIG. 5. which illustrates rescue from serum-induced celldeath by inhibitors of protein and RNA synthesis. The ordinate in FIG. 5represents the number of viable TIG-1 cells after 24 hr incubation. Opencolumns indicate reagent only. Striped columns indicate reagent +1 mMN-acetyl-L-cysteine (NAC). NON, PBS only; CHX, 1 μg/ml cycloheximide;PMC, 1 μgl/ml puromycin.2HCl; EMT, 1 μg/ml emetine.2HCl; L-TRP, 10 mML-tryptophan; D-TRP, 10 mM D-tryptophan; ACT D, 10 μg/ml actinomycin D;ET Br, 25 μg/ml ethidium bromide. TIG-1 cells were seeded in 48-wellmicroplates at a density of 3×10⁴ cells/well in Eagle's MEM containing10% FBS. After 3 days incubation, medium was replaced with 200 μl of FBSplus 50 μl of PBS containing each reagent. After 24 hr, the number ofviable cells was determined by a hemocytometer. All the reagents weretested for rescue from cell death at concentrations ranging from 0.1μg/ml to 10 μg/ml except for D- and L-tryptophan. Data at the mosteffective concentration are shown in this graph. All reagents weretested at concentrations ranging from 0.01 to 10 μg/ml, and in aseparate group 1 mM NAC was added as a positive control. Proteinsynthesis inhibitors CHX, PMC and EMT, and RNA synthesis inhibitors ACTD and ET Br, prevented death of cells in these cultures, suggesting thatserum-induced toxicity requires protein synthesis.

Inhibitors of protein and RNA synthesis protected cells againstserum-induced toxicity similar to thiols, yet they never showed agrowth-promoting effect. Accordingly, these inhibitors may act via amolecular mechanism different from that of thiols. To determine whetherthiols inhibited protein synthesis, L- ³⁵ S!methionine uptake into TIG-1cultured in FBS was measured. A typical result from three independentexperiments is presented in FIG. 6. To develop the data shown in FIG. 6TIG-1 cells were seeded into a 24 well-multiplate at a half-confluency(about 6×10⁴ cells/well), and medium was replaced with FBS containing 20μCi L- ³⁵ S!methionine. After incubation for each indicated time, cellswere lysed and macromolecules were precipitated with TCA. Precipitateswere dissolved in buffer and counted by liquid scintillation. TIG-1cells cultured in FBS showed substantial uptake of L- ³⁵ S!methionine,suggesting that cells vigorously synthesized protein prior to death. L-³⁵ S!methionine uptake into TIG-1 cells in all three experiments wascompletely inhibited by 1 μg/ml CHX and partially inhibited by 5 mML-tryptophan (30-50%). By comparison, 1 mM NAC did not inhibit L- ³⁵S!methionine uptake.

We interpreted these results to indicate that supplemental thiolsprevent cell death by maintaining intracellular thiol levels. We found,however, that acid-soluble thiol content of TIG-1 cells cultured in FBSquickly decreased whether or not the medium was supplemented with thiol(data not shown). Thereafter, total and ethanol-soluble thiol contentwere measured in subsequent experiments. As shown in FIGS. 7a and 7b,both thiol contents were conservative when 1 mM NAC was added to FBS,whereas protein synthesis inhibitor CHX did not affect thiol contenteither total and ethanol-soluble. The following procedure was followedin developing the data plotted in FIGS. 7a and 7b: After several hoursincubation in test medium, cells were lysed in 1% SDS, 0.02% EDTA, 50 mMTris-HCl (pH.8.2). Either the total lysate or the lysate afterprecipitation of macromolecules by x3 volume of ethanol(ethanol-soluble) was dyed with 20 mM DTNB. Absorbance at 415 nm wasmeasured by microplate reader 3550 (Bio Rad). Concentration of thiol wascalculated by comparison to a GSH standard. Thiol content was expressedas SH mol/living cell number. FIGS. 7a and 7b are to be interpreted inconjunction with the following key:

    ______________________________________                                                                                                              10% FBS containing Ea-                                                                        ▴                                                                      FBS + 1 mM NAC;                                       gle's MEM:      ∘                                                                         1 μg/ml CHX;                                 □                                                                        Hanks's balanced solution;                                                                    ▪                                                                           FBS + 1 mM L-TRP.                               Δ                                                                             FBS;                                                                    ______________________________________                                    

Another distinguishing feature of apoptosis, namely, DNA fragmentation,was evaluated by agarose gel electrophoresis of low molecular weight DNAfrom dying cells at various stages of serum toxicity. Living cell ratioswere measured by the method of trypan blue exclusion, as illustrated inFIG. 8. The chart shown in FIG. 8 reflects the result of the followingprocedure: TIG-1 cells were seeded into a 35 mm culture dish at adensity of 2×10⁵ cells/dish. When the density reached 3.5×10⁵cells/dish, medium was replaced with FBS. After the incubation for eachindicated time, cells were harvested with 0.1% trypsin in CMF-PBS, and0.5% trypan blue in PBS was added. Cells that excluded the dyed werecounted as living with a hemocytometer. Results are expressed as themeans (i SE) of four independent determinations. Until 6 hr of the serumtreatment, the cells appeared morphologically viable and were notstained by trypan blue. At that point, viability declined steeply, andall cells were dead by 12 hr. DNA fragments (ladder) appeared at 6 hr ofthe treatment before the plasma membrane breakdown, and they persisteduntil 12 hr. Electrophoresis of DNA fragments of dying cells (not shown)was conducted using 2.6% agarose-gel. The maximal length of the DNAladder was relatively small (<0.7 kbp), and the space between rungs ofthe ladder was slightly smaller than the 0.2 kbp value reported for theDNA ladder usually observed during apoptosis (Kerr et al., 1972; Wyllieet al., 1980; Ellis et al., 1991; Raff, 1992; Eastman, 1993).Nevertheless, DNA fragmentation proceeded morphological change by a fewhours, and RNA and protein synthesis inhibitors prevented cell death,consistent with the view that serum-induced toxicity is a type ofapoptosis.

In the present study, we observed that serum-induced toxicity wasprevented with thiol (SH)-related molecules. Disulfide, D-cysteine orD-cystine, and DTNB were effective in rescuing cells despite the factthat these compounds are not normally present in cells and do notfunction as reducing agents in medium. In addition, dithionite, areducing disulfide, was protective. The findings suggest that thesereagents may promote efficient cellular utilization of SH by dithiolexchange or by a reaction involving chemical reduction. The standardcell culture system is an oxidative environment, where SH is oxidized toform dithiol. SH-compounds like cysteine and glutathione in serum are,therefore, able to form dithiol homo-dimers or mixed disulfides with lowmolecular weight thiols and proteins. In contrast, cells find little usefor mixed disulfides. However, if molecules such as L-cysteine, reducedglutathione, or L-cystine are produced through exchange or reducing,they may be readily used by cells, resulting in promotion ofintracellular SH metabolism.

Intracellular thiol levels were decreased in high concentrations ofserum, and addition of thiols reversed the decline. Thiol content beganto decrease several hours prior to plasma membrane breakdown, and thissuggests that the lowering of thiol content was a causal factor inserum-induced cell death rather than a result. It was not, however, theonly cause of death, because Hank's balanced solution did not inducedeath even though thiol content was reduced as in the case of cellscultured in FBS. Protein synthesis inhibitors prevented cell death, butthey had no effect on intracellular thiol content, suggesting thatprotein synthesis was necessary for serum-induced toxicity.

Taken as a whole, the evidence is consistent with two hypotheses forserum-induced cell death, schematically illustrated in FIGS. 9a and 9b.The first is that a decrease in thiol content directly induces synthesisof proteins required for programmed call death (FIG. 9a). The second isthat a decrease in thiol content affects a variety of intracellularprocesses that occur during cell death including protein synthesis (FIG.9b). Whether either of these hypotheses can be experimentally verifiedremains to be determined. During protein synthesis, de novo RNAsynthesis may not be necessary, since RNA synthesis inhibitors onlypartially protected against death of HeLa cells and low density culturesof TIG-1 cells. High concentrations of L- and D-tryptophan showed equalability to rescue cells. To our knowledge, inhibition of proteinsynthesis by tryptophan has not been reported, yet our preliminary studyshowed that L-tryptophan incompletely inhibited L- ³⁵ S!methionineuptake by cultured TIG-1 cells (FIG. 6). This may indicate that L- andD-tryptophan can act as protein synthesis inhibitors in some cases.

Addition of exogenous thiol compounds maintained the intracellular levelof total thiol, including the protein fraction against depletion byserum-induced toxicity. The level of acid-soluble thiol was, however,not always restored by the addition, whereas that of ethanol solublethiol moderately increased. Ethanol is known to solubilize shortpeptides more efficiently than TCA, and this may be the reason why theethanol soluble thiol level was higher. These results suggest thatsupplemental thiol was predominantly incorporated into protein fractionsthat maintained the total thiol level.

It is possible that thiols contained in protein fractions may act asmodifiers of cysteine residues. For example, some enzymes requiremodification of their cysteine residues by glutathione for activation(Ziegler, 1985). Serum-induced toxicity may reflect such a functionaldisorder caused by a shortage of thiols.

A second major observation was that DNA was cleaved into fragmentsbeginning a few hours before plasma membrane breakdown. Theelectrophoretic behavior of the DNA differed from ordinary apoptosis inthat the fragments were smaller, and sometimes the DNA appeared to berandomly digested, showing a smeared pattern rather than discrete bands.Nevertheless, a characteristic feature of apoptosis was evident, namely,DNA fragmentation prior to membrane disintegration.

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While this invention has been explained with reference to the structuredisclosed herein, it is not confined to the details set forth and thisapplication is intended to cover any modifications and changes as maycome within the scope of the following claims:

What is claimed is:
 1. A method of accelerating serum induced cell deathin a cell system comprising the steps of:providing a medium comprising acytotoxic concentration of serum, said concentration being sufficient toinduce cell death; exposing a population of living cells to said serum;and exposing said cells to glycine in an amount effective to accelerateinduction of death of said cells by said serum.
 2. The method accordingto claim 1, wherein said concentration of serum is at least 60 percent.3. The method according to claim 2, wherein said glycine has aconcentration of 1 mM.
 4. The method according to claim 2, wherein saidliving cells are human fetal lung fibroblasts.
 5. The method accordingto claim 2, wherein said living cells are human epithelioid carcinomacells.
 6. The method according to claim 2, wherein said living cells aremouse melanoma cells.
 7. The method according to claim 1, wherein saidserum comprises whole fetal bovine serum having a concentration of atleast 60 percent.
 8. The method according to claim 7, wherein saidglycine has a concentration of 1 mM.
 9. The method according to claim 7,wherein said living cells are human fetal lung fibroblasts.
 10. Themethod according to claim 7, wherein said living cells are humanepithelioid carcinoma cells.
 11. The method according to claim 7,wherein said living cells are mouse melanoma cells.